1. Field of Invention
This invention relates to photochemical protecting groups or chromophores and more specifically it relates to photorelease of effector molecules by photoreleasing caged compounds.
2. Description of Related Art
Light is an essential tool for studying cells. High photonic fluxes are often required to acquire a distinct signal in fluorescence microcopy, but such high power can also disrupt cells (by heating, singlet oxygen production, etc.), and bleach endo/exogenous chromophores. Photolabile “caged” compounds are inert precursors of bioactive molecules that can be loaded into cells and later released in their active form. Photochemical uncaging of biological signaling molecules typically uses brief bursts of light (near-UV wavelengths for regular, one-photon uncaging, or near-IR light for 2-photon photolysis). This mechanism is highly advantageous in studying the kinetics of important signaling events such as, for example, activation of receptors and ion channels and release of neurotransmitters.
Ionized calcium (Ca2+) is an important second messenger for a wide variety of physiological and biochemical processes such as muscle contraction, neurotransmitter release, ion channel gating, exocytosis, etc. The essential role of Ca2+ release and sequestration in intracellular communication has also been recently highlighted by the growing appreciation of the importance of inositol phospholipid metabolism in signaling. A technique for the controlled, localized, and rapid increase in [Ca2+] would provide a tool which would enable the study of the kinetic, regulatory, and structural mechanisms of such processes. Two approaches to this problem have been taken (see Kaplan, J. H. (1990) Annu. Rev. Physiol. 52, 897-914). The first, developed by Tsien and co-workers, involves reducing the Ca2+-buffering capacity of a BAPTA derivative by decreasing the electron donating capacity of one of the coordinating ligands on illumination following the photoexpulsion of a small molecule from the chelator. This strategy has led to two readily available photosensitive buffers, nitr-5 and nitr-7 (Adams, S. R., Kao, J. P. Y., Grynkiewicz, G., Minta, A., & Tsien, R. Y. (1988) J. Am. Chem. Soc. 110, 3212-3220).
Changes in Ca2+ signaling are observed in various human pathologies such as, for example cancer and neurodegenerative diseases. It has been shown that the spatio-temporal characteristics of Ca2+ signals can regulate the activity of transcription factors and directly affect gene expression.
U.S. Pat. No. 5,446,186 to Ellis-Davies et al. describes an approach to caging Ca2+ and is directed to photosensitive derivatives of chelators with known high affinity for Ca2+, which upon illumination were bifurcated, producing two moieties with known low affinity, thus the bound Ca2+ was released. U.S. Pat. No. 5,446,186 describes a photosensitive Ca2+ chelator, called nitrophenyl-EGTA (NP-EGTA) that binds Ca2+ selectively with high affinity (80 nM), which upon photolysis is bifurcated producing iminodiacetic acid photoproducts with a 12,500-fold lower affinity for Ca2+. This compound possesses the desired properties of Ca2+ selectivity in combination with a rapid high photochemical yield of liberated Ca2+.
DM-nitrophen (U.S. Pat. No. 4,981,985) is a commercially available photosensitive derivative of EDTA has found wide application during the last several years as a photolabile chelator of divalent cations, particularly as caged Ca2+ and caged Mg2+. The distinct advantage of nitr-5 and nitr-7 compared to DM-nitrophen is that they are Ca2+-selective chelators whereas DM-nitrophen has chelation properties similar to EDTA. The comparative advantages of DM-nitrophen are that its Ca2+ affinity is very high before photolysis and very low after photolysis, thus ensuring a good photochemical yield of liberated Ca2+.
U.S. Pat. No. 4,981,985 to Ellis-Davies et al. discloses the synthesis of photolabile chelators for multivalent cations and the method of synthesizing photolabile chelators as EDTA and EGTA derivatives to be used in caging multivalent cations. The molecules chelate the cations forming non-biologically active compounds. Upon irradiation, the chelated cation cleaves with the subsequent cleaved remainders having a substantially lower affinity for the chelated divalent cation. Large amounts of cations are thus rapidly released and the effect of such concentration jumps on the biological system can be accurately studied.
Caging of biomolecules is described by John Corrie in Dynamic Studies in Biology (2005, editors Goeldner and Givens, Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim).
WO04085394A1 to Corrie et al. describes 7-nitroindoline compounds which include a triplet sensitizing group such as substituted or unsubstituted benzophenone group and can be used to cage neurotransmitter effector species.
U.S. Pat. Nos. 5,430,175 and 5,587,509 to Hess et al. describe caged carboxyl compounds and methods of releasing carboxyl compounds in which a 2-alkoxy-5-nitrophenyl photosensitive group blocks a carboxyl function. Preferred compounds are caged neuroactive amino acids (e.g., glutamate and GABA [gamma-aminobutyric acid]) with carboxynitrobenzyl chromophores (CNB) photolyzable by laser pulses at wavelengths above about 350 nm within about 3 microseconds and provide a product quantum yield of greater than about 0.2.
U.S. Pat. No. 5,034,613 to Denk et al. describes a two-photon excitation method that allows accurate spatial discrimination and permits quantification of fluorescence from small volumes whose locations are defined in three dimensions. The two-photon excitation method provides a depth of field resolution comparable to that produced in confocal laser scanning microscopes without the disadvantages of confocal microscopes, previously described. This is especially important in cases where thicker layers of cells are to be studied. Furthermore, the two-photon excitation greatly reduces the background fluorescence.
The photonic flux required for uncaging is typically even more demanding to cell viability than that of fluorescence microscopy. This is because the caging chromophore first deployed for a bio-molecule in 1977 (the ortho-nitrobenzyl photochemical protecting group, Engels & Schlaeger, 1977) and used in the vast majority of uncaging experiments since then, does make efficient use of the incident light. Thus, new caging chromophores that absorb and use light more efficiently so as to be less damaging to cells are desired to address shortcomings of known chromophores, especially for 2-photon excitation uncaging.
All references cited herein are incorporated herein by reference in their entireties.